RESUMO
Due to the rapidly growing number of older people worldwide and the concomitant increase in cardiovascular complications, there is an urgent need for age-related cardiac disease modeling and drug screening platforms. In the present study, we developed a cardiac tissue chip model that incorporates hemodynamic loading and mimics essential aspects of the infarcted aging heart. We induced cellular senescence in H9c2 myoblasts using low-dose doxorubicin treatment. These senescent cells were then used to engineer cardiac tissue fibers, which were subjected to hemodynamic stresses associated with pressure-volume changes in the heart. Myocardial ischemia was modeled in the engineered cardiac tissue via hypoxic treatment. Our results clearly show that acute low-dose doxorubicin treatment-induced senescence, as evidenced by morphological and molecular markers, including enlarged and flattened nuclei, DNA damage response foci, and increased expression of cell cycle inhibitor p16INK4a, p53, and ROS. Under normal hemodynamic load, the engineered cardiac tissues demonstrated cell alignment and retained cardiac cell characteristics. Our senescent cardiac tissue model of hypoxia-induced myocardial infarction recapitulated the pathological disease hallmarks such as increased cell death and upregulated expression of ANP and BNP. In conclusion, the described methodology provides a novel approach to generate stress-induced aging cardiac cell phenotypes and engineer cardiac tissue chip models to study the cardiovascular disease pathologies associated with aging.
Assuntos
Doenças Cardiovasculares , Infarto do Miocárdio , Idoso , Envelhecimento , Doenças Cardiovasculares/complicações , Senescência Celular/genética , Doxorrubicina , Coração , Humanos , Infarto do Miocárdio/patologiaRESUMO
OBJECTIVE AND DESIGN: Phagocytosis and clearance of apoptotic cells are essential for inflammation resolution, efficient wound healing, and tissue homeostasis. MicroRNAs are critical modulators of macrophage polarization and function. The current study aimed to investigate the role of miR-181c-5p in macrophage phagocytosis. MATERIALS AND METHODS: miR-181c-5p was identified as a potential candidate in microRNA screening of RAW264.7 macrophages fed with apoptotic cells. To investigate the role of miR-181c-5p in phagocytosis, the expression of miR-181c-5p was assessed in phagocyting bone marrow-derived macrophages. Phagocytosis efficiency was measured by fluorescence microscopy. Gain- and loss-of-function studies were performed using miR-181c-5p-specific mimic and inhibitor. The expression of the phagocytosis-associated genes and proteins of interest was evaluated by RT2 profiler PCR array and western blotting, respectively. RESULTS: miR-181c-5p expression was significantly upregulated in the phagocyting macrophages. Furthermore, mimic-induced overexpression of miR-181c-5p resulted in the increased phagocytic ability of macrophages. Moreover, overexpression of miR-181c-5p resulted in upregulation of WAVE-2 in phagocyting macrophages, suggesting that miR-181c-5p may regulate cytoskeletal arrangement during macrophage phagocytosis. CONCLUSION: Altogether, our data provide a novel function of miR-181c-5p in macrophage biology and suggest that targeting macrophage miR-181c-5p in injured tissues might improve clearance of dead cells and lead to efficient inflammation resolution.
Assuntos
MicroRNAs , Humanos , Inflamação , Ativação de Macrófagos , Macrófagos/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , FagocitoseRESUMO
DNA damage accrued in induced pluripotent stem cell (iPSC)-derived cardiomyocytes during in vitro culture practices lessens their therapeutic potential. We determined whether DNA-damage-free iPSCs (DdF-iPSCs) can be selected using stabilization of p53, a transcription factor that promotes apoptosis in DNA-damaged cells, and differentiated them into functionally competent DdF cardiomyocytes (DdF-CMs). p53 was activated using Nutlin-3a in iPSCs to selectively kill the DNA-damaged cells, and the stable DdF cells were cultured further and differentiated into CMs. Both DdF-iPSCs and DdF-CMs were then characterized. We observed a significant decrease in the expression of reactive oxygen species and DNA damage in DdF-iPSCs compared with control (Ctrl) iPSCs. Next-generation RNA sequencing and Ingenuity Pathway Analysis revealed improved molecular, cellular, and physiological functions in DdF-iPSCs. The differentiated DdF-CMs had a compact beating frequency between 40 and 60 beats/min accompanied by increased cell surface area. Additionally, DdF-CMs were able to retain the improved molecular, cellular, and physiological functions after differentiation from iPSCs, and, interestingly, cardiac development network was prominent compared with Ctrl-CMs. Enhanced expression of various ion channel transcripts in DdF-CMs implies DdF-CMs are of ventricular CMs and mature compared with their counterparts. Our results indicated that DdF-iPSCs could be selected through p53 stabilization using a small-molecule inhibitor and differentiated into ventricular DdF-CMs with fine-tuned molecular signatures. These iPSC-derived DdF-CMs show immense clinical potential in repairing injured myocardium.NEW & NOTEWORTHY Culture-stress-induced DNA damage in stem cells lessens their performance. A robust small-molecule-based approach, by stabilizing/activating p53, to select functionally competent DNA-damage-free cells from a heterogeneous population of cells is demonstrated. This protocol can be adopted by clinics to select DNA-damage-free cells before transplanting them to the host myocardium. The intact DNA-damage-free cells exhibited with fine-tuned molecular signatures and improved cellular functions. DNA-damage-free cardiomyocytes compared with control expressed superior cardiomyocyte functional properties, including, but not limited to, enhanced ion channel signatures. These DNA-intact cells would better engraft, survive, and, importantly, improve the cardiac function of the injured myocardium.
Assuntos
Diferenciação Celular , Dano ao DNA , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Células Cultivadas , Técnicas de Reprogramação Celular/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Canais Iônicos/genética , Canais Iônicos/metabolismo , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND: Here, we generated human cardiac muscle patches (hCMPs) of clinically relevant dimensions (4 cm × 2 cm × 1.25 mm) by suspending cardiomyocytes, smooth muscle cells, and endothelial cells that had been differentiated from human induced-pluripotent stem cells in a fibrin scaffold and then culturing the construct on a dynamic (rocking) platform. METHODS: In vitro assessments of hCMPs suggest maturation in response to dynamic culture stimulation. In vivo assessments were conducted in a porcine model of myocardial infarction (MI). Animal groups included: MI hearts treated with 2 hCMPs (MI+hCMP, n=13), MI hearts treated with 2 cell-free open fibrin patches (n=14), or MI hearts with neither experimental patch (n=15); a fourth group of animals underwent sham surgery (Sham, n=8). Cardiac function and infarct size were evaluated by MRI, arrhythmia incidence by implanted loop recorders, and the engraftment rate by calculation of quantitative polymerase chain reaction measurements of expression of the human Y chromosome. Additional studies examined the myocardial protein expression profile changes and potential mechanisms of action that related to exosomes from the cell patch. RESULTS: The hCMPs began to beat synchronously within 1 day of fabrication, and after 7 days of dynamic culture stimulation, in vitro assessments indicated the mechanisms related to the improvements in electronic mechanical coupling, calcium-handling, and force generation, suggesting a maturation process during the dynamic culture. The engraftment rate was 10.9±1.8% at 4 weeks after the transplantation. The hCMP transplantation was associated with significant improvements in left ventricular function, infarct size, myocardial wall stress, myocardial hypertrophy, and reduced apoptosis in the periscar boarder zone myocardium. hCMP transplantation also reversed some MI-associated changes in sarcomeric regulatory protein phosphorylation. The exosomes released from the hCMP appeared to have cytoprotective properties that improved cardiomyocyte survival. CONCLUSIONS: We have fabricated a clinically relevant size of hCMP with trilineage cardiac cells derived from human induced-pluripotent stem cells. The hCMP matures in vitro during 7 days of dynamic culture. Transplantation of this type of hCMP results in significantly reduced infarct size and improvements in cardiac function that are associated with reduction in left ventricular wall stress. The hCMP treatment is not associated with significant changes in arrhythmogenicity.
Assuntos
Células Endoteliais/transplante , Células-Tronco Pluripotentes Induzidas/transplante , Infarto do Miocárdio/cirurgia , Miocárdio/patologia , Miócitos Cardíacos/transplante , Miócitos de Músculo Liso/transplante , Regeneração , Transplante de Células-Tronco/métodos , Engenharia Tecidual/métodos , Animais , Diferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/patologia , Miócitos de Músculo Liso/patologia , Recuperação de Função Fisiológica , Regeneração/genética , Sus scrofa , Fatores de Tempo , Alicerces Teciduais , Transplante Heterólogo , Função Ventricular Esquerda , Remodelação VentricularRESUMO
The role of p53 transactivation domain (p53-TAD), a multifunctional and dynamic domain, on DNA repair and retaining DNA integrity in human induced pluripotent stem cells (hiPSCs) has never been studied. p53-TAD was knocked out in iPSCs using CRISPR/Cas9 and was confirmed by DNA sequencing. p53-TAD knockout (KO) cells were characterized by accelerated proliferation, decreased population doubling time, and unaltered Bcl-2, Bcl-2-binding component 3, insulin-like growth factor 1 receptor, and Bax and altered Mdm2, p21, and p53-induced death domain transcript expression. In p53-TAD KO cells, the p53-regulated DNA repair proteins xeroderma pigmentosum group A, DNA polymerase H, and DNA-binding protein 2 expression were found to be reduced compared with p53 wild-type cells. Exposure to a low dose of doxorubicin (Doxo) induced similar DNA damage and DNA damage response (DDR) as measured by RAD50 and MRE11 expression, checkpoint kinase 2 activation, and γH2A.X recruitment at DNA strand breaks in both cell groups, indicating that silence of p53-TAD does not affect the DDR mechanism upstream of p53. After removal of Doxo, p53 wild-type hiPSCs underwent DNA repair, corrected their damaged DNA, and restored DNA integrity. Conversely, p53-TAD KO hiPSCs did not undergo complete DNA repair and failed to restore DNA integrity. More importantly, continuous culture of p53-TAD KO hiPSCs underwent G2/M cell cycle arrest and expressed the cellular senescent marker p16INK4a. Our data clearly show that silence of the TAD of p53 did not affect DDR but affected the DNA repair process, implying the crucial role of p53-TAD in maintaining DNA integrity. Therefore, activating p53-TAD domain using small molecules may promote DNA repair and integrity of cells and prevent cellular senescence.
Assuntos
Reparo do DNA , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Hidrolases Anidrido Ácido/metabolismo , Pontos de Checagem do Ciclo Celular , Células Cultivadas , Quinase do Ponto de Checagem 2/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Doxorrubicina/toxicidade , Instabilidade Genômica , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Proteína Homóloga a MRE11/metabolismo , Domínios Proteicos , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genéticaRESUMO
Vascular endothelial growth factor (VEGF) is a well-characterized proangiogenic cytokine that has been shown to promote neovascularization in hearts of patients with ischemic heart disease but can also lead to adverse effects depending on the dose and mode of delivery. We investigated whether prolonged exposure to a low dose of VEGF could be achieved by encapsulating VEGF in polylactic coglycolic acid nanoparticles and whether treatment with VEGF-containing nanoparticles improved cardiac function and protected against left ventricular remodeling in the hearts of mice with experimentally induced myocardial infarction. Polylactic coglycolic acid nanoparticles with a mean diameter of ~113 nm were generated via double emulsion and loaded with VEGF; the encapsulation efficiency was 53.5 ± 1.7% (107.1 ± 3.3 ng VEGF/mg nanoparticles). In culture, VEGF nanoparticles released VEGF continuously for at least 31 days, and in a murine myocardial infarction model, VEGF nanoparticle administration was associated with significantly greater vascular density in the peri-infarct region, reductions in infarct size, and improvements in left ventricular contractile function 4 wk after treatment. Thus, our study provides proof of principle that nanoparticle-mediated delivery increases the angiogenic and therapeutic potency of VEGF for the treatment of ischemic heart disease. NEW & NOTEWORTHY Vascular endothelial growth factor (VEGF) is a well-characterized proangiogenic cytokine but has a short half-life and a rapid clearance rate. When encapsulated in nanoparticles, VEGF was released for 31 days and improved left ventricular function in infarcted mouse hearts. These observations indicate that our new platform increases the therapeutic potency of VEGF.
Assuntos
Indutores da Angiogênese/administração & dosagem , Infarto do Miocárdio/tratamento farmacológico , Nanopartículas , Neovascularização Fisiológica/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/administração & dosagem , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacos , Indutores da Angiogênese/química , Animais , Células Cultivadas , Preparações de Ação Retardada , Modelos Animais de Doenças , Portadores de Fármacos , Composição de Medicamentos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Contração Miocárdica/efeitos dos fármacos , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Recuperação de Função Fisiológica , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/químicaRESUMO
The microenvironment of native heart tissue may be better replicated when cardiomyocytes are cultured in three-dimensional clusters (i.e., spheroids) than in monolayers or as individual cells. Thus, we differentiated human cardiac lineage-induced pluripotent stem cells in cardiomyocytes (hiPSC-CMs) and allowed them to form spheroids and spheroid fusions that were characterized in vitro and evaluated in mice after experimentally induced myocardial infarction (MI). Synchronized contractions were observed within 24 h of spheroid formation, and optical mapping experiments confirmed the presence of both Ca2+ transients and propagating action potentials. In spheroid fusions, the intraspheroid conduction velocity was 7.0 ± 3.8 cm/s on days 1- 2 after formation, whereas the conduction velocity between spheroids increased significantly ( P = 0.003) from 0.8 ± 1.1 cm/s on days 1- 2 to 3.3 ± 1.4 cm/s on day 7. For the murine MI model, five-spheroid fusions (200,000 hiPSC-CMs/spheroid) were embedded in a fibrin patch and the patch was transplanted over the site of infarction. Later (4 wk), echocardiographic measurements of left ventricular ejection fraction and fractional shortening were significantly greater in patch-treated animals than in animals that recovered without the patch, and the engraftment rate was 25.6% or 30% when evaluated histologically or via bioluminescence imaging, respectively. The exosomes released from the spheroid patch seemed to increase cardiac function. In conclusion, our results established the feasibility of using hiPSC-CM spheroids and spheroid fusions for cardiac tissue engineering, and, when fibrin patches containing hiPSC-CM spheroid fusions were evaluated in a murine MI model, the engraftment rate was much higher than the rates we have achieved via the direct intramyocardial injection. NEW & NOTEWORTHY Spheroids fuse in culture to produce structures with uniformly distributed cells. Furthermore, human cardiac lineage-induced pluripotent stem cells in cardiomyocytes in adjacent fused spheroids became electromechanically coupled as the fusions matured in vitro, and when the spheroids were combined with a biological matrix and administered as a patch over the infarcted region of mouse hearts, the engraftment rate exceeded 25%, and the treatment was associated with significant improvements in cardiac function via a paracrine mechanism, where exosomes released from the spheroid patch.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Infarto do Miocárdio/terapia , Miócitos Cardíacos/transplante , Esferoides Celulares/transplante , Animais , Sinalização do Cálcio , Células Cultivadas , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Contração Miocárdica , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Esferoides Celulares/metabolismo , Transplante de Células-Tronco/métodosRESUMO
Human induced pluripotent stem cell (hiPSC)-derived cardio-myocytes (hiPSC-CMs) hold great promise for cardiovascular disease modeling and regenerative medicine. However, these cells are both structurally and functionally -immature, primarily due to their differentiation into cardiomyocytes occurring under static culture which only reproduces biomolecular cues and ignores the dynamic hemo-dynamic cues that shape early and late heart development during cardiogenesis. To evaluate the effects of hemodynamic stimuli on hiPSC-CM maturation, we used the biomimetic cardiac tissue model to reproduce the hemodynamics and pressure/volume changes associated with heart development. Following 7 days of gradually increasing stimulation, we show that hemodynamic loading results in (a) enhanced alignment of the cells and extracellular matrix, (b) significant increases in genes associated with physiological hypertrophy, (c) noticeable changes in sarcomeric organization and potential changes to cellular metabolism, and (d) a significant increase in fractional shortening, suggestive of a positive force frequency response. These findings suggest that culture of hiPSC-CMs under conditions that accurately reproduce hemodynamic cues results in structural orga-nization and molecular signaling consistent with organ growth and functional maturation.
Assuntos
Técnicas de Cultura de Células/instrumentação , Diferenciação Celular , Hemodinâmica , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Biomimética/instrumentação , Biomimética/métodos , Técnicas de Cultura de Células/métodos , Linhagem Celular , Desenho de Equipamento , Humanos , Miócitos Cardíacos/ultraestrutura , Sarcômeros/ultraestruturaRESUMO
Studies of myocardial aging are complex and the mechanisms involved in the deterioration of ventricular performance and decreased functional reserve of the old heart remain to be properly defined. We have studied a colony of beagle dogs from 3 to 14 yr of age kept under a highly regulated environment to define the effects of aging on the myocardium. Ventricular, myocardial, and myocyte function, together with anatomical and structural properties of the organ and cardiomyocytes, were evaluated. Ventricular hypertrophy was not observed with aging and the structural composition of the myocardium was modestly affected. Alterations in the myocyte compartment were identified in aged dogs, and these factors negatively interfere with the contractile reserve typical of the young heart. The duration of the action potential is prolonged in old cardiomyocytes contributing to the slower electrical recovery of the myocardium. Also, the remodeled repolarization of cardiomyocytes with aging provides inotropic support to the senescent muscle but compromises its contractile reserve, rendering the old heart ineffective under conditions of high hemodynamic demand. The defects in the electrical and mechanical properties of cardiomyocytes with aging suggest that this cell population is an important determinant of the cardiac senescent phenotype. Collectively, the delayed electrical repolarization of aging cardiomyocytes may be viewed as a critical variable of the aging myopathy and its propensity to evolve into ventricular decompensation under stressful conditions.
Assuntos
Potenciais de Ação , Envelhecimento/fisiologia , Miócitos Cardíacos/fisiologia , Função Ventricular , Animais , Cães , Feminino , Hemodinâmica , MasculinoAssuntos
Dano ao DNA , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/transplante , Animais , Apoptose , Diferenciação Celular , Separação Celular , Estenose Coronária/terapia , Vasos Coronários , Modelos Animais de Doenças , Genes p53 , Sobrevivência de Enxerto , Imidazóis/farmacologia , Células-Tronco Pluripotentes Induzidas/química , Ligadura , Camundongos , Camundongos SCID , Miócitos Cardíacos/citologia , Piperazinas/farmacologiaRESUMO
BACKGROUND: The efficacy of bypass surgery in patients with ischemic cardiomyopathy is not easily predictable; preoperative clinical conditions may be similar, but the outcome may differ significantly. We hypothesized that the growth reserve of cardiac stem cells (CSCs) and circulating cytokines promoting CSC activation are critical determinants of ventricular remodeling in this patient population. METHODS AND RESULTS: To document the growth kinetics of CSCs, population-doubling time, telomere length, telomerase activity, and insulin-like growth factor-1 receptor expression were measured in CSCs isolated from 38 patients undergoing bypass surgery. Additionally, the blood levels of insulin-like growth factor-1, hepatocyte growth factor, and vascular endothelial growth factor were evaluated. The variables of CSC growth were expressed as a function of the changes in wall thickness, chamber diameter and volume, ventricular mass-to-chamber volume ratio, and ejection fraction, before and 12 months after surgery. A high correlation was found between indices of CSC function and cardiac anatomy. Negative ventricular remodeling was not observed if CSCs retained a significant growth reserve. The high concentration of insulin-like growth factor-1 systemically pointed to the insulin-like growth factor-1-insulin-like growth factor-1 receptor system as a major player in the adaptive response of the myocardium. hepatocyte growth factor, a mediator of CSC migration, was also high in these patients preoperatively, as was vascular endothelial growth factor, possibly reflecting the vascular growth needed before bypass surgery. Conversely, a decline in CSC growth was coupled with wall thinning, chamber dilation, and depressed ejection fraction. CONCLUSIONS: The telomere-telomerase axis, population-doubling time, and insulin-like growth factor-1 receptor expression in CSCs, together with a high circulating level of insulin-like growth factor-1, represent a novel biomarker able to predict the evolution of ischemic cardiomyopathy following revascularization.
Assuntos
Ponte de Artéria Coronária , Isquemia Miocárdica/patologia , Isquemia Miocárdica/cirurgia , Miocárdio/patologia , Células-Tronco/patologia , Idoso , Biomarcadores/sangue , Proliferação de Células , Células Cultivadas , Citocinas/sangue , Feminino , Seguimentos , Fator de Crescimento de Hepatócito/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Isquemia Miocárdica/sangue , Valor Preditivo dos Testes , Receptor IGF Tipo 1/sangue , Células-Tronco/ultraestrutura , Telomerase/fisiologia , Telômero/ultraestrutura , Resultado do Tratamento , Fator A de Crescimento do Endotélio Vascular/sangueRESUMO
BACKGROUND: Aging negatively impacts on the function of resident human cardiac progenitor cells (hCPCs). Effective regeneration of the injured heart requires mobilization of hCPCs to the sites of damage. In the young heart, signaling by the guidance receptor EphA2 in response to the ephrin A1 ligand promotes hCPC motility and improves cardiac recovery after infarction. METHODS AND RESULTS: We report that old hCPCs are characterized by cell-autonomous inhibition of their migratory ability ex vivo and impaired translocation in vivo in the damaged heart. EphA2 expression was not decreased in old hCPCs; however, the elevated level of reactive oxygen species in aged cells induced post-translational modifications of the EphA2 protein. EphA2 oxidation interfered with ephrin A1-stimulated receptor auto-phosphorylation, activation of Src family kinases, and caveolin-1-mediated internalization of the receptor. Cellular aging altered the EphA2 endocytic route, affecting the maturation of EphA2-containing endosomes and causing premature signal termination. Overexpression of functionally intact EphA2 in old hCPCs corrected the defects in endocytosis and downstream signaling, enhancing cell motility. Based on the ability of phenotypically young hCPCs to respond efficiently to ephrin A1, we developed a novel methodology for the prospective isolation of live hCPCs with preserved migratory capacity and growth reserve. CONCLUSIONS: Our data demonstrate that the ephrin A1/EphA2 pathway may serve as a target to facilitate trafficking of hCPCs in the senescent myocardium. Importantly, EphA2 receptor function can be implemented for the selection of hCPCs with high therapeutic potential, a clinically relevant strategy that does not require genetic manipulation of stem cells.
Assuntos
Células-Tronco Adultas/fisiologia , Envelhecimento/fisiologia , Movimento Celular/fisiologia , Miocárdio/citologia , Receptor EphA2/metabolismo , Transdução de Sinais/fisiologia , Adulto , Células-Tronco Adultas/citologia , Idoso , Células Cultivadas , Endocitose/fisiologia , Efrina-A1/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Receptor EphA2/genética , Regeneração/fisiologia , Transferrina/metabolismoRESUMO
BACKGROUND: Little is known about the function of inositol 1,4,5-trisphosphate receptors (IP3Rs) in the adult heart experimentally. Moreover, whether these Ca(2+) release channels are present and play a critical role in human cardiomyocytes remains to be defined. IP3Rs may be activated after Gαq-protein-coupled receptor stimulation, affecting Ca(2+) cycling, enhancing myocyte performance, and potentially favoring an increase in the incidence of arrhythmias. METHODS AND RESULTS: IP3R function was determined in human left ventricular myocytes, and this analysis was integrated with assays in mouse myocytes to identify the mechanisms by which IP3Rs influence the electric and mechanical properties of the myocardium. We report that IP3Rs are expressed and operative in human left ventricular myocytes. After Gαq-protein-coupled receptor activation, Ca(2+) mobilized from the sarcoplasmic reticulum via IP3Rs contributes to the decrease in resting membrane potential, prolongation of the action potential, and occurrence of early afterdepolarizations. Ca(2+) transient amplitude and cell shortening are enhanced, and extrasystolic and dysregulated Ca(2+) elevations and contractions become apparent. These alterations in the electromechanical behavior of human cardiomyocytes are coupled with increased isometric twitch of the myocardium and arrhythmic events, suggesting that Gαq-protein-coupled receptor activation provides inotropic reserve, which is hampered by electric instability and contractile abnormalities. Additionally, our findings support the notion that increases in Ca(2+) load by IP3Rs promote Ca(2+) extrusion by forward-mode Na(+)/Ca(2+) exchange, an important mechanism of arrhythmic events. CONCLUSIONS: The Gαq-protein/coupled receptor/IP3R axis modulates the electromechanical properties of the human myocardium and its propensity to develop arrhythmias.
Assuntos
Potenciais de Ação/fisiologia , Sinalização do Cálcio/fisiologia , Insuficiência Cardíaca/fisiopatologia , Receptores de Inositol 1,4,5-Trifosfato/fisiologia , Miócitos Cardíacos/fisiologia , Adulto , Animais , Arritmias Cardíacas/fisiopatologia , Células Cultivadas , Feminino , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/fisiologia , Insuficiência Cardíaca/genética , Ventrículos do Coração/citologia , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Contração Miocárdica/fisiologia , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Retículo Sarcoplasmático/fisiologia , Transdução de Sinais/fisiologiaRESUMO
This article has been withdrawn at the request of editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.
Assuntos
Doenças Cardiovasculares/terapia , Congressos como Assunto/tendências , Relatório de Pesquisa/tendências , Transplante de Células-Tronco/tendências , Engenharia Tecidual/tendências , Alabama , Animais , Humanos , Transplante de Células-Tronco/métodos , Células-Tronco , Engenharia Tecidual/métodosRESUMO
Nonsurgical bleeding occurs in a significant proportion of patients implanted with continuous-flow ventricular assist devices (CF-VADs) and is associated with nonphysiologic flow with diminished pulsatility. An in vitro vascular pulse perfusion model seeded with adult human aortic endothelial cells (HAECs) was used to identify biomarkers sensitive to changes in pulsatility. Diminished pulsatility resulted in an ~45% decrease in von Willebrand factor (vWF) levels from 9.80 to 5.32 ng/ml (n = 5, p < 0.05) and a threefold increase in angiopoietin-2 (ANGPT-2) levels from 775.29 to 2471.93 pg/ml (n = 5, p < 0.05) in cultured HAECs. These changes are in agreement with evaluation of patient blood samples obtained pre-CF-VAD implant and 30-day postimplant: a decrease in plasma vWF level by 50% from ~45.59 to ~22.49 µg/ml (n = 15, p < 0.01) and a 64% increase in plasma ANGPT-2 level from 7,073 to 11,615 pg/ml (n = 8, p < 0.05). This study identified vWF and ANGPT-2 as highly sensitive to changes in pulsatility, in addition to interleukin-6 (IL-6), IL-8, and tumor necrosis-α (TNF-α). These biomarkers may help determine the optimal level of pulsatility and help identify patients at high risk of nonsurgical bleeding.
Assuntos
Coração Auxiliar , Doenças de von Willebrand , Adulto , Humanos , Fator de von Willebrand , Células Endoteliais , Coração Auxiliar/efeitos adversos , Angiopoietina-2 , Hemorragia/etiologia , Biomarcadores , Doenças de von Willebrand/etiologiaRESUMO
Discovery of the molecular targets of traditional medicine and its chemical footprints can validate the use of such medicine. In the present report, we investigated the effect of ursolic acid (UA), a pentacyclic triterpenoid found in rosemary and holy basil, on apoptosis induced by TRAIL. We found that UA potentiated TRAIL-induced apoptosis in cancer cells. In addition, UA also sensitized TRAIL-resistant cancer cells to the cytokine. When we investigated the mechanism, we found that UA down-regulated cell survival proteins and induced the cell surface expression of both TRAIL receptors, death receptors 4 and 5 (DR4 and -5). Induction of receptors by UA occurred independently of cell type. Gene silencing of either receptor by small interfering RNA reduced the apoptosis induced by UA and the effect of TRAIL. In addition, UA also decreased the expression of decoy receptor 2 (DcR2) but not DcR1. Induction of DRs was independent of p53 because UA induced DR4 and DR5 in HCT116 p53(-/-) cells. Induction of DRs, however, was dependent on JNK because UA induced JNK, and its pharmacologic inhibition abolished the induction of the receptors. The down-regulation of survival proteins and up-regulation of the DRs required reactive oxygen species (ROS) because UA induced ROS, and its quenching abolished the effect of the terpene. Also, potentiation of TRAIL-induced apoptosis by UA was significantly reduced by both ROS quenchers and JNK inhibitor. In addition, UA was also found to induce the expression of DRs, down-regulate cell survival proteins, and activate JNK in orthotopically implanted human colorectal cancer in a nude mouse model. Overall, our results showed that UA potentiates TRAIL-induced apoptosis through activation of ROS and JNK-mediated up-regulation of DRs and down-regulation of DcR2 and cell survival proteins.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , MAP Quinase Quinase 4/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Triterpenos/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima/efeitos dos fármacos , Animais , Antineoplásicos Fitogênicos/agonistas , Apoptose/genética , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Sinergismo Farmacológico , Humanos , MAP Quinase Quinase 4/genética , Camundongos , Camundongos Nus , Transplante de Neoplasias , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Triterpenos/agonistas , Receptores Chamariz do Fator de Necrose Tumoral/agonistas , Receptores Chamariz do Fator de Necrose Tumoral/genética , Receptores Chamariz do Fator de Necrose Tumoral/metabolismo , Proteína Supressora de Tumor p53/genética , Regulação para Cima/genética , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Ácido UrsólicoRESUMO
Most anticancer drugs have their origin in traditional medicinal plants. We describe here a flavone, 5,3'-dihydroxy-3,6,7,8,4'-pentamethoxyflavone (PMF), from the leaves of the Thai plant Gardenia obtusifolia, that has anti-inflammatory and anticancer potential. Because the nuclear factor-κB (NF-κB) pathway is linked to inflammation and tumorigenesis, we investigated the effect of PMF on this pathway. We found that PMF suppressed NF-κB activation induced by inflammatory agents, tumor promoters, and carcinogens. This suppression was not specific to the cell type. Although PMF did not directly modify the ability of NF-κB proteins to bind to DNA, it inhibited IκBα (inhibitory subunit of NF-κB) kinase, leading to suppression of phosphorylation and degradation of IκBα, and suppressed consequent p65 nuclear translocation, thus abrogating NF-κB-dependent reporter gene expression. Suppression of the NF-κB cell signaling pathway by the flavone led to the inhibition of expression of NF-κB-regulated gene products that mediate inflammation (cyclooxygenase-2), survival (XIAP, survivin, Bcl-xL, and cFLIP), proliferation (cyclin D1), invasion (matrix metalloproteinase-9), and angiogenesis (vascular endothelial growth factor). Suppression of antiapoptotic gene products by PMF correlated with the enhancement of apoptosis induced by tumor necrosis factor-α and the chemotherapeutic agents cisplatin, paclitaxel, and 5-flurouracil. Overall, our results indicate that PMF suppresses the activation of NF-κB and NF-κB-regulated gene expression, leading to the enhancement of apoptosis. This is the first report to demonstrate that this novel flavone has anti-inflammatory and anticancer effects by targeting the IKK complex.